This application is a 371 of PCT/EP01/07941 filed Jul. 10, 2001, now WO 02/04420 Jan. 17, 2002.
This invention relates to organic compounds, their preparation and their use as pharmaceuticals.
In one aspect, the invention provides compounds of formula 
in free or salt form, where
Ar1 is phenyl substituted by one or more halogen atoms,
Ar2 is phenyl or naphthyl which is unsubstituted or substituted by one or more substituents selected from halogen, cyano, hydroxy, nitro, C1-C8-alkyl, C1-C8-haloalkyl, C1-C8-alkoxy or C1-C8-alkoxycarbonyl,
R1 is hydrogen or C1-C8-alkyl optionally substituted by hydroxy, C1-C8-alkoxy, acyloxy, xe2x80x94N(R2)R3, halogen, carboxy, C1-C8-alkoxycarbonyl, xe2x80x94CON(R4)R5 or by a monovalent cyclic organic group,
R2 and R3 are each independently hydrogen or C1-C8-alkyl, or R2 is hydrogen and R3 is acyl or xe2x80x94SO2R6, or R2 and R3 together with the nitrogen atom to which they are attached denote a 5- or 6-membered heterocyclic group,
R4 and R5 are each independently hydrogen or C1-C8-alkyl, or R4 and R5 together with the nitrogen atom to which they are attached denote a 5- or 6-membered heterocyclic group,
R6 is C1-C8-alkyl, C1-C8-haloalkyl, or phenyl optionally substituted by C1-C8-alkyl, and n is 1, 2,3 or 4,
with the proviso that when Ar1 is p-chlorophenyl and R1 is hydrogen, Ar2 is not phenyl or p-nitrophenyl.
Terms used in the specification have the following meanings:
xe2x80x9cC1-C8-alkylxe2x80x9d as used herein denotes straight chain or branched C1-C8-alkyl, which may be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, straight or branched pentyl, straight or branched hexyl, straight or branched heptyl, or straight or branched octyl. Preferably, C1-C8-alkyl is C1-C4-alkyl.
xe2x80x9cC1-C8-alkoxyxe2x80x9d as used herein denotes straight chain or branched C1-C8-alkoxy which may be, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, straight or branched pentoxy, straight or branched hexyloxy, straight or branched heptyloxy, or straight or branched octyloxy. Preferably, C1-C8-alkoxy is C1-C4-alkoxy.
xe2x80x9cC1-C8-haloalkylxe2x80x9d as used herein denotes C1-C8-alkyl as hereinbefore defined substituted by one or more halogen atoms, preferably one, two or three halogen atoms.
xe2x80x9cAcylxe2x80x9d as used herein denotes alkylcarbonyl, for example C1-C8-alkylcarbonyl where C1-C8-alkyl may be one of the C1-C8-alkyl groups hereinbefore mentioned, optionally substituted by one or more halogen atoms; cycloalkylcarbonyl, for example C3-C8-cycloalkylcarbonyl where C3-C8-cycloalkyl may be, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl; 5- or 6-membered heterocyclylcarbonyl having one or two hetero atoms selected from nitrogen, oxygen and sulfur in the ring, such as furylcarbonyl or pyridylcarbonyl; arylcarbonyl, for example C6-C10-arylcarbonyl such as benzoyl; or aralkylcarbonyl, for example C6 to C10-aryl-C1-C4-alkylcarbonyl such as benzylcarbonyl or phenylethylcarbonyl. Preferably acyl is C1-C4-alkylcarbonyl.
xe2x80x9cAcyloxyxe2x80x9d as used herein denotes alkylcarbonyloxy, for example C1-C8-alkylcarbonyloxy where C1-C8-alkyl may be one of the C1-C8-alkyl groups hereinbefore mentioned, optionally substituted by one or more halogen atoms; cycloalkylcarbonyloxy, for example C3-C8-cycloalkylcarbonyloxy where C3-C8-cycloalkyl may be, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl; 5- or 6-membered heterocyclylcarbonyloxy having one or two hetero atoms selected from nitrogen, oxygen and sulfur in the ring, such as furylcarbonyloxy or pyridylcarbonyloxy; arylcarbonyloxy, for example C6-C10-arylcarbonyloxy such as benzoyloxy; or aralkylcarbonyloxy, for example C6 to C10-aryl-C1-C4-alkylcarbonyloxy such as benzylcarbonyloxy or phenylethylcarbonyloxy. Preferably acyloxy is C1-C4-alkylcarbonyloxy.
xe2x80x9cHalogenxe2x80x9d as used herein may be fluorine, chlorine, bromine or iodine; preferably it is fluorine, chlorine or bromine.
In Ar1, the phenyl group may be substituted by one, two or three, preferably one or two halogen atoms, preferably selected from fluorine and chlorine atoms. When there is one halogen substituent, it is preferably para to the indicated carbonyl group. When there are two or three halogen substituents, preferably one is para to the indicated carbonyl group and at least one of the others is ortho to the indicated carbonyl group.
Ar2 as substituted phenyl may, for example, be substituted by one, two, three, four or five, preferably by one, two or three, of the abovementioned substituents. Ar2 may be, for example, monosubstituted phenyl in which the substituent, preferably halogen, cyano, nitro or C1-C4-alkoxy, is preferably ortho or meta to the indicated xe2x80x94CHxe2x95x90CHxe2x80x94 group. Ar2 may alternatively be, for example, disubstituted phenyl in which the substituents are preferably selected from halogen, cyano, hydroxy, nitro, C1-C4-alkoxy, C1-C4-alkyl and C1-C4-haloalkyl, especially two halogen substituents (same or different halogen), two C1-C4-alkoxy groups, two C1-C4-alkyl groups, two C1-C4-haloalkyl groups, one halogen and one cyano, one halogen and one C1-C4-alkoxy, one halogen and one nitro, one halogen and one hydroxy, one halogen and one C1-C4-haloalkyl, one cyano and one C1-C4-alkoxy, one hydroxy and one C1-C4-alkyl, or one hydroxy and one C1-C4-alkoxy group. Ar2 may alternatively be, for example, trisubstituted phenyl in which the substituents are preferably selected from halogen, hydroxy, C1-C4-alkoxy and C1-C4-alkoxycarbonyl, especially three halogen substituents (same or two or three different halogens), or two C1-C4-alkoxy and one halogen, hydroxy or C1-C4-alkoxycarbonyl. Ar2 may alternatively be, for example, penta-substituted phenyl in which the substituents are preferably halogen, especially fluorine. Especially preferred groups Ar2 are cyanophenyl, particularly meta-cyanophenyl, and disubstituted phenyl where one substituent is C1-C4-alkoxy, preferably ortho to the xe2x80x94CHxe2x95x90CHxe2x80x94 group, and the other, preferably para to the C1-C4-alkoxy group, is C1-C4-alkoxy, halogen, cyano or C1-C4-alkyl.
R1 as optionally substituted C1-C8-alkyl is preferably optionally substituted C1-C4-alkyl, especially C1-C4-alkyl or substituted methyl or ethyl. When R1 is substituted by a cyclic organic group, the latter may be a carbocyclic or heterocyclic group, for example a C3-C15-carbocyclic group or a 5- to 7-membered heterocyclic group having one or more, preferably one, two or three, ring hetero atoms selected from nitrogen, oxygen and sulfur. The C3-C15-carbocyclic group may be, for example, a cycloaliphatic group having 3 to 8 carbon atoms, preferably C5- or C6-cycloalkyl such as cyclopentyl, methylcyclopentyl or cyclohexyl. The C3-C15-carbocyclic group may alternatively be, for example, a C6-C15 aromatic group, such as phenyl, which is unsubstituted or substituted by C1-C8-alkyl, C1-C8-alkoxy, halogen, cyano, xe2x80x94CON(R4)R5, xe2x80x94SO2N(R4)R5 or C1-C8-alkylsulfonylamino where R4 and R5 are a hereinbefore defined. The heterocyclic group may have one nitrogen, oxygen or sulfur atom in the ring or it may have two nitrogens, or one oxygen and one or two nitrogens, or one sulfur and one or two nitrogens in the ring. The heterocyclic group is preferably a heterocyclic aromatic group, especially a 5- or 6-membered heterocyclic group such as furyl, imidazolyl, thiazolyl or pyridyl. In especially preferred compounds, R1 is C1-C4-alkyl substituted by hydroxy, phenyl, or a 5-or 6-membered heterocyclic aromatic group having one or two ring hetero atoms selected from nitrogen, oxygen and sulfur.
Preferred compounds of formula I in free or salt form include those in which
Ar1 is phenyl substituted by fluorine or chlorine para to the indicated carbonyl group and optionally further substituted by halogen ortho to the indicated carbonyl group,
Ar2 is phenyl monosubstituted by a substituent selected from halogen, cyano, nitro and C1-C4-alkoxy, phenyl substituted by two substituents, which may be the same or different, selected from halogen, cyano, hydroxy, C1-C4-alkoxy, C1-C4-alkyl, C1-C4-haloalkyl and nitro, or phenyl substituted by three substituents, which may be the same or different, selected from halogen, hydroxy, C1-C4-alkoxy and C1-C4-alkoxycarbonyl,
R1 is hydrogen, C1-C4-alkyl or C1-C4-alkyl substituted by hydroxy, C3-C8-cycloalkyl, phenyl, C1-C4-alkylsulfonylamino-substituted phenyl or a 5- or 6-membered heterocyclic aromatic group having one or more ring hetero atoms selected from nitrogen, oxygen and sulfur, and n is 1 or 2.
Further preferred compounds of formula I in free or salt form include those in which
Ar1 is phenyl substituted by fluorine or chlorine para to the indicated carbonyl group,
Ar2 is phenyl substituted ortho to the indicated xe2x80x94CHxe2x95x90CHxe2x80x94 group by C1-C4-alkoxy and para to the C1-C4-alkoxy group by cyano, halogen or C1-C4-alkoxy,
R1 is C1-C4-alkyl substituted by hydroxy, phenyl, C1-C4-alkylsulfonylamino-substituted phenyl or a 5- or 6-membered heterocyclic aromatic group having one or two ring hetero atoms selected from nitrogen, oxygen and sulfur, and n is 1.
The compounds represented by formula I are capable of forming acid addition salts, particularly pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable acid addition salts of the compound of formula I include those of inorganic acids, for example, hydrohalic acids such as hydrofluoric acid, hydrochloric acid, hydrobromic acid or hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid; and organic acids, for example aliphatic monocarboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid and butyric acid, aliphatic hydroxy acids such as lactic acid, citric acid, tartaric acid or malic acid, dicarboxylic acids such as maleic acid or succinic acid, aromatic carboxylic acids such as benzoic acid, p-chlorobenzoic acid, diphenylacetic acid or triphenylacetic acid, aromatic hydroxy acids such as o-hydroxybenzoic acid, p-hydroxybenzoic acid, 1-hydroxynaphthalene-2-carboxylic acid or 3-hydroxynaphthalene-2-carboxylic acid, and sulfonic acids such as methanesulfonic acid or benzenesulfonic acid. These salts may be prepared from compounds of formula I by known salt-forming procedures.
Compounds of formula I which contain acidic, e.g. carboxyl, groups, are also capable of forming salts with bases, in particular pharmaceutically acceptable bases such as those well known in the art; suitable such salts include metal salts, particularly alkali metal or alkaline earth metal salts such as sodium, potassium, magnesium or calcium salts, or salts with ammonia or pharmaceutically acceptable organic amines or heterocyclic bases such as ethanolamines, benzylamines or pyridine. These salts may be prepared from compounds of formula I by known salt-forming procedures.
When R1 is other than hydrogen, the carbon atom to which R1 is attached in formula I is asymmetric, in which case the compounds exist in individual optically active isomeric forms or as mixtures thereof, e.g. as racemic or diastereomeric mixtures. The invention embraces both individual optically active R and S isomers as well as mixtures, e.g. racemic or diastereomeric mixtures, thereof.
Specific especially preferred compounds of the invention are those described hereinafter in the Examples, particularly those of Examples 4, 9, 10, 15, 18, 19, 20, 21, 23, 24, 25, 28, 29, 30, 37, 38,40, 42, 43, 44 and 45.
The invention also provides a process for the preparation of compounds of formula I which comprises
(i) (A) reacting a compound of formula 
with a compound of formula 
or an amide-forming derivative thereof, where Ar1, Ar2, R1 and n are as hereinbefore defined, or
(B) reacting a compound of formula m, or an amide forming derivative thereof, with a compound of formula 
where Ar1, R1 and n are as hereinbefore defined and Z denotes a solid phase substrate chemically linked to the indicated nitrogen atom, and detaching the resulting product from the substrate to replace Z by hydrogen; and
(ii) recovering the product in free or salt form.
In process variant (A), the compound of formula II may be in free or salt form. Process variant (A) may be effected using known methods, for example by reacting a compound of formula II with an acid halide, particularly acid chloride, of the acid of formula III using known amide-forming procedures. Conveniently, the compound of formula II in free or salt form is reacted with a free carboxylic acid of formula III, for example using known procedures, such as reaction in the presence of a tertiary amine and a peptide coupling agent such as a phosphonium salt, 2-(1H benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate or diisopropylcarbodiimide; this reaction may be carried out in an inert organic solvent, for example a halohydrocarbon such as dichloromethane; the reaction temperature is conveniently from 0 to 40xc2x0 C., preferably ambient temperature.
In another method of effecting process variant (A), a compound of formula II, preferably in salt form, is reacted with an amide-forming derivative of the acid of formula III which is a thioester of formula 
where Ar2 is as hereinbefore defined. The reaction may be carried using known procedures or analogously as described hereinafter in the Examples. It may be effected in the presence of a tertiary base such as N-methylmorpholine. It is conveniently effected in an organic solvent, preferably an alcohol such as ethanol. The reaction temperature may be, for example, from 30 to 60xc2x0 C., conveniently from 40 to 50xc2x0 C.
Process variant (B) may be effected using known methods, for example by reacting the substrate-bound compound with the free acid under known peptide coupling conditions, for example in the presence of a tertiary amine and a peptide coupling agent such as those mentioned above. The reaction may be effected in an inert organic solvent such as dimethylformamide (DMF). Suitable reaction temperatures are from 0 to 40xc2x0 C., e.g. 15 to 25xc2x0 C. The product may be detached from the substrate in a known manner, for example, where the N atom is linked to a CH2 of a benzyl group in Z, by treatment with trifluoroacetic acid.
Compounds of formula III are either available commercially or may be prepared by known methods. Compounds of formula IIIA may be obtained by reaction of an acid of formula III with 2,2xe2x80x2-dibenzothiazolyl disulfide in the presence of triphenylphosphine and a tertiary base such as N-methylmorpholine, e.g. as described in the Examples.
Compounds of formula II may be prepared by reacting a compound of formula 
with a compound of formula 
where Ar1, R1 and n are as hereinbefore defined, with the proviso that when R1 contains a reactive functional group such as a hydroxy group, the reactive group may be in protected form, for example a hydroxy group protected as a tert-butoxy group, R7 is hydrogen or an amine-protective group, for example a tert-butoxycarbonyl group, and X is halogen and, where R7 is a protective group, replacing R7 in the product by hydrogen, and, where R1 in the product contains a protected functional group, replacing the protecting group by hydrogen. When R7 is hydrogen, reaction between a compound of formula V and a salt of a compound of formula VI may be effected by the procedures described in U.S. Pat. No. 4,559,349. When R7 is a protective group, reaction between compounds of formulae V and VI may be effected using known methods, for example in the presence of a tertiary organic base such as triethylamine or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), conveniently in an inert organic solvent, for example a polar solvent such as dimethylformamide, the reaction temperature suitably being from 0 to 40xc2x0 C., preferably ambient temperature. Replacement of a protective group R7 by hydrogen may be effected using known procedures; for example, where R7 is tert-butoxycarbonyl, by treatment with a carboxylic acid such as trifluoroacetic acid. Replacement of a protecting group in R1 may be affected using known procedures, for example, when R1 contains a hydroxy group protected as an ether group, such as tert-butoxy, by treatment with HBr in a carboxylic acid such as acetic acid; when R7 is a protective group, this treatment also replaces R7 by hydrogen. Compounds of formulae V and VI are known or may be prepared by known procedures.
Where reference is made herein to protected functional groups or to protecting groups, the protecting groups may be chosen in accordance with the nature of the functional group, for example as described in Protective Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, John Wiley and Sons Inc, Second Edition, 1991, which reference also describes procedures suitable for replacement of the protecting groups by hydrogen.
Compounds of formula II may also be prepared by reacting a compound of formula V with a compound of formula 
where R1, R7 and n are as hereinbefore defined and a reducing agent such as sodium cyanoborohydride or sodium triacetoxyborohydride, for example using known reductive amination procedures, conveniently in an inert organic solvent, for example an ether such as tetrahydrofuran (THF), the reaction temperature suitably being from 0 to 40xc2x0 C., and, where R7 is a protective group, replacing it by hydrogen. Compounds of formula VII are known or may be prepared by known procedures.
Compounds of formula II where R1 is hydroxymethyl may also be prepared by reacting a compound of formula V with (R)4-formyl-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester, of formula 
and a reducing agent such as sodium triacetoxyborohydride, for example under conditions described above for reaction of compounds of formulae V and VII, and reacting the product with a suitable reagent to cleave the oxazolidine ring and replace the nitrogen-bound ester group by hydrogen, for example hydrogen chloride in ethanol or dioxane as described hereinafter in the Examples, in which case the compound of formula II is obtained as a hydrochloride salt. The reaction product of the compounds of formulae V and VIIa may, e.g. where it is desired to improve enantiometric purity, be treated with an optically active acid such as di-O,O-benzoyl-L-tartaric acid before cleavage of the oxazolidine ring. The compound of formula VIIa may be prepared as described by A D Campbell et al, Synthesis 1707-1709 (1998) or G Ageno et al, Tetrahedron 51, 8121-8134 (1995).
Compounds of formula II where R1 is C1-C8-alkoxymethyl or acyloxymethyl can be prepared by appropriate etherification or acylation of compounds of formula II where R1 is hydroxymethyl.
Compounds of formula IV may be prepared by reacting a compound of formula V with a compound of formula 
where R1, Z and n are as hereinbefore defined, for example using known procedures such as reaction in an inert organic solvent such as DMF in the presence of a tertiary amine, conveniently at a temperature of 40 to 60xc2x0 C. Compounds of formula VIII may be prepared by reaction of a compound of formula 
where R1, Z and n are as hereinbefore defined, with iodine, for example using known procedures such as reaction in an inert organic solvent such as a mixture of THF and acetonitrile in the presence of a triarylphosphine and imidazole, conveniently at a temperature of 10 to 40xc2x0 C. Compounds of formula IX may be prepared by reaction of a compound of formula 
where where R1 and n are as hereinbefore defined, with a solid phase substrate Z having a group, such as an aldehyde group, reactive with amino. Such solid phase substrates, including modified resins, particularly modified polystyrene resins, are commercially available. Compounds of formula X are known or may be prepared by known methods.
Compounds of formula I in free form may be converted into salt form, and vice versa, in a conventional manner. The compounds in free or salt form can be obtained in the form of hydrates or solvates containing a solvent used for crystallization. Compounds of formula I can be recovered from reaction mixtures and purified in a conventional manner. Isomers, such as enantiomers, may be obtained in a conventional manner, e.g. by fractional crystallization or asymmetric synthesis from correspondingly asymmetrically substituted, e.g. optically active, starting materials.
Compounds of formula I in free or pharmaceutically acceptable salt form, hereinafter referred to alternatively as agents of the invention, are useful as pharmaceuticals. Accordingly the invention also provides a compound of formula I in free or pharmaceutically acceptable salt form for use as a pharmaceutical. The agents of the invention act as CCR-3 receptor antagonists, thereby inhibiting the infiltration and activation of inflammatory cells, particularly eosinophils, and inhibiting allergic response. The inhibitory properties of agents of the invention can be demonstrated in the following assay:
CCR-3 Binding Assay
In this assay the effect of agents of the invention on the binding of human eotaxin to human CCR-3 is determined. Recombinant cells expressing human CCR-3 are captured by wheatgerm agglutinin (WGA) polyvinyltoluidene (PVT) SPA beads (available from Amersham), through a specific interaction between the WGA and carbohydrate residues of glycoproteins on the surface of the cells. [125I]-human eotaxin (available from Amersham) binds specifically to CCR-3 receptors bringing the [125I]-human eotaxin in close proximity to the SPA beads. Emitted xc3xa2-particles from the [125I]-human eotaxin excite, by its proximity, the fluorophore in the beads and produce light. Free [125I]-human eotaxin in solution is not in close proximity to the scintillant and hence does not produce light. The scintillation count is therefore a measure of the extent to which the test compound inhibits binding of the eotaxin to the CCR-3.
Preparation of Assay Buffer: 5.96 g HEPES and 7.0 g sodium chloride are dissolved in distilled water and 1M aqueous CaCl2 (1 mL) and 1M aqueous MgCl2 (5 mL) are added. The pH is adjusted to 7.6 with NaOH and the solution made to a final volume of 1 L using distilled water. 5 g bovine serum albumin and 0.1 g sodium azide are then dissolved in the solution and the resulting buffer stored at 4xc2x0 C. A Complete(trademark) protease inhibitor cocktail tablet (available from Boehringer) is added per 50 mL of the buffer on the day of use.
Preparation of Homogenisation Buffer: Tris-base (2.42 g) is dissolved in distilled water, the pH of the solution is adjusted to 7.6 with hydrochloric acid and the solution is diluted with distilled water to a final volume of 1L. The resulting buffer is stored at 4xc2x0 C. A Complete(trademark) protease inhibitor cocktail tablet is added per 50 mL of the buffer on the day of use.
Preparation of membranes: Confluent rat basophil leukemia (RBL-2H3) cells stably expressing CCR3 are removed from tissue culture flasks using enzyme-free cell dissociation buffer and resuspended in phosphate-buffered saline. The cells are centrifuged (800 g, 5 minutes), the pellet resuspended in ice-cold homogenisation buffer using 1 mL homogenisation buffer per gram of cells and incubated on ice for 30 minutes. The cells are homogenised on ice with 10 strokes in a glass mortar and pestle. The homogenate is centrifuged (800 g, 5 minutes, 4xc2x0 C.), the supernatant further centrifuged (48,000 g, 30 minutes, 4xc2x0 C.) and the pellet redissolved in Homogenisation Buffer containing 10% (v/v) glycerol. The protein content of the membrane preparation is estimated by the method of Bradford (Anal.Biochem. (1976) 72:248) and aliquots are snap frozen and stored at xe2x88x9280xc2x0 C. The assay is performed in a final volume of 250 xcexcL per well of an Optiplate (ex Canberra Packard). To selected wells of the Optiplate are added 50 xcexcL of solutions of a test compound in Assay Buffer containing 5% DMSO (concentrations from 0.1 nM to 10 xcexcM). To determine total binding, 50 xcexcL of the Assay Buffer containing 5% DMSO is added to other selected wells. To determine non-specific binding, 50 xcexcL of 100 nM human eotaxin (ex RandD Systems) in Assay Buffer containing 5% DMSO is added to further selected wells. To all wells are added 50 xcexcL [125I]-Human eotaxin (ex Amersham) in Assay Buffer containing 5% DMSO at a concentration of 250 pM (to give a final concentration of 50 pM per well), 50 xcexcL of WGA-PVT SPA beads in Assay Buffer (to give a final concentration of 1.0 mg beads per well) and 100 xcexcL of the membrane preparation at a concentration of 100 xcexcg protein in Assay Buffer (to give a final concentration of 10 xcexcg protein per well). The plate is then incubated for 4 hours at room temperature. The plate is sealed using TopSeal-S (ex Canberra Packard) according to the manufacturer""s instructions. The resulting scintillations are counted using a Canberra Packard TopCount, each well being counted for 1 minute. The concentration of test compound at which 50% inhibition occurs (IC50) is determined from concentration-inhibition curves in a conventional manner.
The compounds of the Examples hereinbelow have IC50 values below 1 xcexcM in the above assay. For instance, the compounds of Examples 1, 2, 4, 7, 9, 13, 20, 23, 25, 28, 30, 38, 40, 43 and 44 have IC50(nM) values of 125, 68, 13, 15, 5, 26, 8, 10, 11, 2, 13, 14, 6, 22 and 25 respectively.
Most of the compounds of the Examples exhibit selectivity for inhibition of CCR-3 binding relative to inhibition of binding of the alpha-1 adrenergic receptor. The inhibitory properties of agents of the invention on binding of the alpha-1 adrenergic receptor can be determined in the following assay:
Cerebral cortices from male Sprague-Dawley rats (175-200 g) are dissected and homogenised in 10 volumes of ice cold 0.32 M sucrose (containing 1 mM MgCl2 dihydrate and 1 mM K2HPO4) with a glass/teflon homogeniser. The membranes are centrifuged at 1000xc3x97g for 15 min., the pellet discarded and the centrifugation repeated. The supernatants are pooled and centrifuged at 18,000xc3x97g for 15 min. The pellet is osmotically shocked in 10 volumes of water and kept on ice for 30 min. The suspension is centrifuged at 39,000xc3x97g for 20 min., resuspended in Krebs-Henseleit buffer pH 7.4 (1.17 mM MgSO4 anhydrous, 4.69 mM KCl, 0.7 mM K2HPO4 anhydrous, 0.11M NaCl, 11 mM D-glucose and 25 mM NaHCO3) containing 20 mM Tris, and kept for 2 days at xe2x88x9220xc2x0 C. The membranes are then thawed at 20-23xc2x0 C., washed three times with Krebs-Henseleit buffer by centrifugation at 18,000xc3x97g for 15 min., left overnight at 4xc2x0 C. and washed again three times. The final pellet is resuspended with a glass/teflon homogeniser in 125 mL/100 membranes in the same buffer. A sample is taken to determine the protein concentration (using the Bradford Assay with gamma globulin as the standard) and the remainder aliquoted and stored at xe2x88x9280xc2x0 C.
The resulting membranes are subjected to a radioligand binding assay. The assay is conducted in triplicate using 96 well plates containing [125I]-HEAT (Amersham) (40 pM, Kd: 58.9xc2x118.7 pM), unlabelled test compound and membrane (57.1 xcexcg/mL) to yield a final volume of 250 xcexcl (assay buffer containing 50 mM Tris-base and 0.9% (w/v) NaCl, pH 7.4). The plates are incubated at 37xc2x0 C. for 60 min., after which rapid vacuum filtration over Whatman GF/C 96 well filter plates is carried out. Each plate is then washed three times with 10 ml of ice cold assay buffer using a Brandel Cell harvester (Gaithersburg, Md.). Following drying of the plates for 3 h. at 50xc2x0 C., 40 xcexcL of Microscint 20 is added to each well, the plates incubated at room temperature for a further 20 min. and the retained radioactivity quantified in a Packard Topcount NXT scintillation counter.
Stock solutions of test compounds are dissolved initially in 100% DMSO and diluted with assay buffer to the required concentrations to yield 1% (v/v) DMSO.
The concentration of test compound at which 50% inhibition occurs (IC50) is determined from concentration-inhibition curves in a conventional manner. Compounds of Examples 1, 2, 4, 7, 9, 13, 20, 23, 25, 28, 30, 38, 40, 43 and 44 have IC50(nM) values of 210, 221, 94, 48, 58, 53, 89, 131, 387, 72, 121, 1519, 215, 356 and 331 in this assay.
Having regard to their inhibition of binding of CCR-3, agents of the invention are useful in the treatment of conditions mediated by CCR-3, particularly inflammatory or allergic conditions. Treatment in accordance with the invention may be symptomatic or prophylactic.
Accordingly, agents of the invention are useful in the treatment of inflammatory or obstructive airways diseases, resulting, for example, in reduction of tissue damage, bronchial hyperreactivity, remodelling or disease progression. Inflammatory or obstructive airways diseases to which the present invention is applicable include asthma of whatever type or genesis including both intrinsic (non-allergic) asthma and extrinsic (allergic) asthma, mild asthma, moderate asthma, severe asthma, bronchitic asthma, excercise-induced asthma, occupational asthma and asthma induced following bacterial or viral infection. Treatment of asthma is also to be understood as embracing treatment of subjects, e.g. of less than 4 or 5 years of age, exhibiting wheezing symptoms and diagnosed or diagnosable as xe2x80x9cwheezy infantsxe2x80x9d, an established patient category of major medical concern and now often identified as incipient or early-phase asthmatics. (For convenience this particular asthmatic condition is referred to as xe2x80x9cwheezy-infant syndromexe2x80x9d.)
Prophylactic efficacy in the treatment of asthma will be evidenced by reduced frequency or severity of symptomatic attack, e.g. of acute asthmatic or bronchoconstrictor attack, improvement in lung function or improved airways hyperreactivity. It may further be evidenced by reduced requirement for other, symptomatic therapy, i.e. therapy for or intended to restrict or abort symptomatic attack when it occurs, for example anti-inflammatory (e.g. corticosteroid) or bronchodilatory. Prophylactic benefit in asthma may in particular be apparent in subjects prone to xe2x80x9cmorning dippingxe2x80x9d. xe2x80x9cMorning dippingxe2x80x9d is a recognised asthmatic syndrome, common to a substantial percentage of asthimatics and characterised by asthma attack, e.g. between the hours of about 4 to 6 am, i.e. at a time normally substantially distant form any previously administered symptomatic asthma therapy.
Other inflammatory or obstructive airways diseases and conditions to which the present invention is applicable include acute lung injury (ALI), acute/adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary, airways or lung disease (COPD, COAD or COLD), including chronic bronchitis or dyspnea associated therewith, emphysema, as well as exacerbation of airways hyperreactivity consequent to other drug therapy, in particular other inhaled drug therapy. The invention is also applicable to the treatment of bronchitis of whatever type or genesis including, e.g., acute, arachidic, catarrhal, croupus, chronic or phthinoid bronchitis. Further inflammatory or obstructive airways diseases to which the present invention is applicable include pneumoconiosis (an inflammatory, commonly occupational, disease of the lungs, frequently accompanied by airways obstruction, whether chronic or acute, and occasioned by repeated inhalation of dusts) of whatever type or genesis, including, for example, aluminosis, anthracosis, asbestosis, chalicosis, ptilosis, siderosis, silicosis, tabacosis and byssinosis.
Having regard to their anti-inflammatory activity, in particular in relation to inhibition of eosinophil activation, agents of the invention are also useful in the treatment of eosinophil related disorders, e.g. eosinophilia, in particular eosinophil related disorders of the airways (e.g. involving morbid eosinophilic infiltration of pulmonary tissues) including hypereosinophilia as it effects the airways and/or lungs as well as, for example, eosinophil-related disorders of the airways consequential or concomitant to Lxc3x6ffler""s syndrome, eosinophilic pneumonia, parasitic (in particular metazoan) infestation (including tropical eosinophilia), bronchopulmonary aspergillosis, polyarteritis nodosa (including Churg-Strauss syndrome), eosinophilic granuloma and eosinophil-related disorders affecting the airways occasioned by drug-reaction.
Agents of the invention are also useful in the treatment of inflammatory or allergic conditions of the skin, for example psoriasis, contact dermatitis, atopic dermatitis, alopecia areata, erythema multiforma, dermatitis herpetiformis, scleroderma, vitiligo, hypersensitivity angiitis, urticaria, bullous pemphigoid, lupus erythematosus, pemphisus, epidermolysis bullosa acquisita, and other inflammatory or allergic conditions of the skin.
Agents of the invention may also be used for the treatment of other diseases or conditions, in particular diseases or conditions having an inflammatory component, for example, treatment of diseases and conditions of the eye such as conjunctivitis, keratoconjunctivitis sicca, and vernal conjunctivitis, diseases affecting the nose including allergic rhinitis, e.g. atrophic, chronic, or seasonal rhinitis, inflammatory conditions of the gastrointestinal tract, for example inflammatory bowel disease such as ulcerative colitis and Crohn""s disease, diseases of the bone and joints including rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and systemic sclerosis, and other diseases such as athersclerosis, multiple sclerosis, diabetes (type I), myasthenia gravis, hyper IgE syndrome and acute and chronic allograft rejection, e.g. following transplantation of heart, kidney, liver, lung or bone marrow.
The effectiveness of an agent of the invention in inhibiting inflammatory conditions, for example in inflammatory airways diseases, may be demonstrated in an animal model, e.g. a mouse or rat model, of airways inflammation or other inflammatory conditions, for example as described by Szarka et al, J. Immunol. Methods (1997) 202:49-57; Renzi et al, Am. Rev. Respir. Dis. (1993) 148:932-939; Tsuyuki et al., J. Clin. Invest. (1995) 96:2924-2931; and Cernadas et al (1999) Am. J. Respir. Cell Mol. Biol. 20:1-8.
The agents of the invention are also useful as co-therapeutic agents for use in combination with other drug substances such as anti-inflammatory, bronchodilatory or antihistamine drug substances, particularly in the treatment of obstructive or inflammatory airways diseases such as those mentioned hereinbefore, for example as potentiators of therapeutic activity of such drugs or as a means of reducing required dosaging or potential side effects of such drugs. An agent of the invention may be mixed with the other drug substance in a fixed pharmaceutical composition or it may be administered separately, before, simultaneously with or after the other drug substance. Such anti-inflammatory drugs include steroids, in particular glucocorticosteroids such as budesonide, beclamethasone, fluticasone, ciclesonide or mometasone, LTB4 antagonists such as those described in U.S. Pat. No. 5,451,700, LTD4 antagonists such as montelukast and zafirlukast, dopamine receptor agonists such as cabergoline, bromocriptine, ropinirole and 4-hydroxy-7-[2-[[2-[[3-(2-phenylethoxy)propyl]sulfonyl]ethyl]-amino]ethyl]-2(3H)-benzothiazolone and pharmaceutically acceptable salts thereof (the hydrochloride being Viozan(copyright)xe2x80x94AstraZeneca), and PDE4 inhibitors such as Ariflo(copyright) (GlaxoSmith Kline), Roflumilast (Byk Gulden), V-11294A (Napp), BAY19-8004 (Bayer), SCH-351591 (Schering-Plough), and PD189659 (Parke-Davis). Such bronchodilatory drugs include anticholinergic or antimuscarinic agents, in particular ipratropium bromide, oxitropium bromide and tiotropium bromide, and beta-2 adrenoceptor agonists such as salbutamol, terbutaline, salmeterol and, especially, formoterol and pharmaceutically acceptable salts thereof, and compounds (in free or salt or solvate form) of formula I of PCT International Publication No. WO00/75114, which document is incorporated herein by reference, preferably compounds of the Examples thereof, especially a compound of formula 
and pharmaceutically acceptable salts thereof. Co-therapeutic antihistamine drug substances include cetirizine hydrochloride, acetaminophen, clemastine fumarate, promethazine, loratidine, desloratidine, diphenhydramine and fexofenadine hydrochloride. Combinations of agents of the invention and steroids, beta-2 agonists, PDE4 inhibitors or LTD4 antagonists may be used, for example, in the treatment of COPD or, particularly, asthma. Combinations of agents of the invention and anticholinergic or antimuscarinic agents, PDE4 inhibitors, dopamine receptor agonists or LTB4 antagonists may be used, for example, in the treatment of asthma or, particularly, COPD.
Other useful combinations of agents of the invention with anti-inflammatory drugs are those with other anatagonists of chemokine receptors, e.g. CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9 and CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, particularly CCR-5 antagonists such as Schering-Plough antagonists SC-351125, SCH-55700 and SCH-D, Takeda antagonists such as N-[[4-[[[6,7-dihydro-2-(4-methylphenyl)-5H-benzocyclohepten-8-yl]carbonyl]amino]phenyl]-methyl]tetrahydro-N,N-dimethyl-2H-pyran-4-aminium chloride (TAK-770), and CCR-5 antagonists described in U.S. Pat. No. 6,166,037 (particularly claims 18 and 19), WO00/66558 (particularly claim 8), and WO00/66559 (particularly claim 9).
In accordance with the foregoing, the invention also provides a method for the treatment of a condition mediated by CCR-3, for example an inflammatory or allergic condition, particularly an inflammatory or obstructive airways disease, which comprises administering to a subject, particularly a human subject, in need thereof an effective amount of a compound of formula I in a free or pharmaceutically acceptable salt form as hereinbefore described. In another aspect the invention provides the use of a compound of formula I, in free or pharmaceutically acceptable salt form, as hereinbefore described for the manufacture of a medicament for the treatment of a condition mediated by CCR-3, for example an inflammatory or allergic condition, particularly an inflammatory or obstructive airways disease.
The agents of the invention may be administered by any appropriate route, e.g. orally, for example in the form of a tablet or capsule; parenterally, for example intravenously; by inhalation, for example in the treatment of inflammatory or obstructive airways disease; intranasally, for example in the treatment of allergic rhinitis; topically to the skin, for example in the treatment of atopic dermatitis; or rectally, for example in the treatment of inflammatory bowel disease.
In a further aspect, the invention also provides a pharmaceutical composition comprising as active ingredient a compound of formula I in free or pharmaceutically acceptable salt form, optionally together with a pharmaceutically acceptable diluent or carrier therefor. The composition may contain a co-therapeutic agent such as an anti-inflammatory or bronchodilatory drug as hereinbefore described. Such compositions may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus oral dosage forms may include tablets and capsules. Formulations for topical administration may take the form of creams, ointments, gels or transdermal delivery systems, e.g. patches. Compositions for inhalation may comprise aerosol or other atomizable formulations or dry powder formulations.
The invention includes (A) an agent of the invention in inhalable form, e.g. in an aerosol or other atomisable composition or in inhalable particulate, e.g. micronised form, (B) an inhalable medicament comprising an agent of the invention in inhalable form; (C) a pharmaceutical product comprising such an agent of the invention in inhalable form in association with an inhalation device; and (D) an inhalation device containing an agent of the invention in inhalable form.
Dosages of agents of the invention employed in practising the present invention will of course vary depending, for example, on the particular condition to be treated, the effect desired and the mode of administration. In general, suitable daily dosages for administration by inhalation are of the order of 0.01 to 30 mg/kg while for oral administration suitable daily doses are of the order of 0.01 to 100 mg/kg.